子囊霉素菌株合成酶的构建及调控表达机制

Rate-limiting enzymes of ascomycin biosynthesis in Streptomyces. hygroscopicus var. ascomyceticus are focused in this research and systematically identified under full understanding of the ascomycin synthetic mechanism which simulated and revealed by high reliable and accurate genome-scale dynamic metabolic model with the validation of unsteady 13C-labeling experiments. The structures of rate-limiting enzymes are decoupled and abstracted with the synthetic biological modularization method, to analysis the mechanisms of the function motifs of catalytic activity, coenzyme recognition and conformational adjustment, then recombination, rearrangement and fusion of motifs are employed for the design of synthetic enzymes. Construction researches of synthetic enzymes following the line “artificial parts-function models-synthetic pathways” would be progressed, and then the test, feedback and verification system with fingerprinting of enzyme catalysis would also be established to give a deep study in the match rules between synthetic enzymes, therefore, high efficient synthetic enzymes are eventually constructed. The chassis is engineered with the method of “minimal genome”, and the detecting and analysis system of “omics” are employed to reveal the regulation and expression mechanisms between the synthetic pathway and the chassis, for obtaining a high-efficient stable ascomycin-producing strain.

针对吸水链霉菌子囊亚种原始菌株，开发非稳态13C标记实验检测体系，实验验证获得高精准基因组尺度动态代谢网络模型及模拟，系统解析子囊霉素合成代谢机制，挖掘子囊霉素合成限速酶/酶系；运用合成生物学的模块化设计思想，解耦和抽提限速酶/酶系的蛋白质结构特征，解析催化活性、辅酶识别、构象调节等功能基序机制，通过基序人工重组、重排、融合等方法完成合成酶结构与功能的科学设计；开展“人工元件—功能模块化—合成途径”的多层次合成酶构建研究，建立酶催化指纹图谱检测与反馈验证体系，研究合成酶催化和匹配规律，完成高效合成酶构建；采用“最小基因组”技术构建底盘细胞，通过“组学”检测技术，深入分析和揭示子囊霉素合成途径与底盘细胞之间的调控表达机制，获得高效稳定的子囊霉素人工合成菌株。

针对吸水链霉菌子囊亚种原始菌株，开发非稳态13C标记实验检测体系，实验验证获得高精准基因组尺度动态代谢网络模型及模拟，系统解析子囊霉素合成代谢机制，挖掘子囊霉素合成限速酶/酶系；运用合成生物学的模块化设计思想，解耦和抽提限速酶/酶系的蛋白质结构特征，解析催化活性、辅酶识别、构象调节等功能基序机制，通过基序人工重组、重排、融合等方法完成合成酶结构与功能的科学设计；开展“人工元件—功能模块化—合成途径”的多层次合成酶构建研究，建立酶催化指纹图谱检测与反馈验证体系，研究合成酶催化和匹配规律，完成高效合成酶构建；采用“最小基因组”技术构建底盘细胞，通过“组学”检测技术，深入分析和揭示子囊霉素合成途径与底盘细胞之间的调控表达机制。在项目执行中，按照研究内容和技术路线，取得了重要结果，达到了项目结题要求，对子囊霉素菌珠全基因组代谢网络模型和模拟、异源合成与表达均具有重要的科学价值和潜在的经济效益。